The disulfide bond as a chemical tool in cyclic peptide antibiotics: engineering disulfide polymyxins and murepavadin

多药耐药(MDR)病原体的增加已成为全球关注的问题。据最近估计，2019年全球有495万人死于耐药细菌感染。其中127万人的死亡直接归因于抗菌素耐药性(AMR)。在欧洲，欧洲疾病预防和控制中心(ECDC)报告称，抗药性细菌感染导致3.3万人死亡(欧洲经济区数据)。同样，在美国，美国疾病控制与预防中心估计，每年发生超过280万例耐药感染，导致超过3.5万人死亡。世界卫生组织和疾控中心已将耐碳青霉烯类鲍曼不动杆菌、肠杆菌科(包括大肠杆菌和肺炎克雷伯菌)和铜绿假单胞菌(也是多重耐药菌株)等革兰氏阴性病原体确定为紧急或严重威胁。关于与耐药性有关的死亡人数，六种主要病原体(大肠杆菌、金黄色葡萄球菌、肺炎克雷伯菌、肺炎链球菌、鲍曼不动杆菌和铜绿假单胞菌)导致2019年约929,000人死于AMR。特别是，耐粘菌素、碳青霉烯类或多药耐药铜绿假单胞菌在2015年欧盟和欧洲经济区因感染抗生素耐药细菌而导致的死亡和残疾调整寿命年的影响最大。因此，这些铜绿假单胞菌引起的感染的治疗在该领域显然是一个未得到满足的医疗需求。在这方面，抗菌肽，特别是那些环状、非核糖体生物合成(或核糖体合成和翻译后修饰)的抗菌肽，提供了作为潜在药物进行探索的机会。在这一类中，我们发现了众所周知的临床使用药物，如达托霉素和多粘菌素(多粘菌素B和多粘菌素E/粘菌素，用于前药甲氧基甲酸酯)或新化合物，如达罗巴汀或替克生。达托霉素主要用于治疗革兰氏阳性金黄色葡萄球菌引起的感染，而多粘菌素已成为治疗多重耐药(MDR)革兰氏阴性菌(GNB)引起的感染的最后治疗选择。当多粘菌素的肾毒性和神经毒性导致没有其他治疗选择时，多粘菌素被用作最后的抗生素。因此，没有这种不良反应的新型多粘菌素的出现将是治疗由多重耐药(MDR)革兰氏阴性细菌引起的感染的一大进步。该项目提出了一种创新的化学工具，可应用于已知的环肽抗生素，包括那些正在临床使用或处于临床开发阶段的环肽抗生素，以降低其肾毒性。设计的基本原理是保持抗生素的整体结构以保持抗菌活性，而在肽骨架中存在化学工具将通过在抗生素最终在肾脏中积累时通过解毒酶来促进初始代谢。该项目遵循概念验证计划，涉及制备模型化合物所需的化学，体外和体内测试以评估活性和低毒性，并估计治疗窗口。最后，还提出了在膜水平上验证设计假设和作用机理的试验。

The increase of multidrug-resistant (MDR) pathogens has become a matter of global concern. It has been recently estimated that 4.95 million people died worldwide due to drug-resistant bacterial infections in 2019. Of those, 1.27 million deaths were directly attributed to antimicrobial resistance (AMR). In Europe, the European Centre for Disease Prevention and Control (ECDC) has reported that antibiotic-resistant bacterial infections caused 33,000 deaths (data for the European Economic Area). Similarly, in the US, the CDC estimates that more than 2.8 million antibiotic-resistant infections take place annually with more than 35,000 dead people as a consequence. The WHO and CDC have identified Gram-negative pathogens such as carbapenem-resistant strains of Acinetobacter baumannii, Enterobacteriaceae (which including Escherichia coli and Klebsiella pneumoniae), and Pseudomonas aeruginosa (also multi-drug resistant strains) as urgent or serious threats. Regarding the number of deaths associated with resistance, six leading pathogens (E. coli, S. aureus, K. pneumoniae, Streptococcus pneumoniae, A. baumannii, and P. aeruginosa) were responsible for ca 929,000 deaths attributable to AMR in 2019.In particular, colistin-resistant, carbapenem-resistant, or multidrug-resistant P. aeruginosa shows some of the highest impact related to deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European Economic Area in 2015. Hence, the treatment of these P. aeruginosa caused infections is a clear unmet medical need in the field. In this regards, antimicrobial peptides, particularly those that are cyclic, non-ribosomally biosynthesized (or ribosomally synthesized and post-translationally modified) offer an opportunity to be explored as potential drugs. In this class we find well-known clinically-used drugs such as daptomycin and polymyxins (polymyxin B and polymyxin E/colistin, used in the prodrug form colistimethate) or novel compounds such as darobactin or teixobactin. Daptomycin is mostly indicated to treat Gram-positive Staphylococcus aureus caused infections, whereas polymyxins have become a last resort therapeutic option to treat multi-drug resistant (MDR) Gram-negative bacteria (GNB) caused infections. Polymyxins are used as last resort antibiotics when no other therapeutic option is available due to their nephrotoxicity and neurotoxicity. Hence, the availability of novel polymyxins devoid of such adverse effects would be a great advance for the treatment of infections caused by multi-drug resistant (MDR) Gram-negative bacteria. The project presents an innovative chemical tool to be applied to known cyclic peptide antibiotics, including those in clinical use or in a clinical development phase, to reduce their nephrotoxicity. The rationale of the design consists of maintaining the overall structure of the antibiotic to preserve the antibacterial activity while the presence of the chemical tool within the peptide backbone would facilitate the initial metabolization by detoxifying enzymes upon eventual accumulation of the antibiotic in the kidney. The project follows a proof-of-concept scheme involving the necessary chemistry to prepare the model compounds, the in vitro and in vivo assays to assess activity and low toxicity, and estimation the therapeutic window. Finally, tests to prove the design hypothesis and the mechanism of action at the membrane level are also proposed.